Mononuclear non-heme iron active sites are present in a range of enzymes involved in a variety of biological functions requiring O2. These include the lipoxygenases (LOs, fatty acid hydroperoxidation), bleomycin (BLM, DNA cleavage), intra- and extradiol dioxygenases (degradation of aromatic rings), pterin-dependent hydroxylases (amino acid metabolism), and alpha- ketoglutarate (alpha-KG)-dependent enzymes (hydroxylation and ring closure). Substrate activation by FeIII and O2 activation by FeII sites are found for different enzymes, and key intermediates have been observed. Much less is known about these enzymes relative to heme systems as the non-heme iron centers are less spectroscopically accessible. Research goals have been to develop new spectroscopic methods for the investigation of non-heme iron active sites to obtain molecular level insight into their catalytic mechanisms and define differences in active site geometric and electronic structure which relate to differences in O2 and substrate reactivity. These studies should also contribute significantly toward elucidating the relation of non-heme to heme iron sites and the nature of O2 activation in a non-heme environment. Studies have emphasized magnetic circular dichroism combined with other excited state spectroscopic methods to probe the geometric and electronic structure of non-heme FeII and FeIII sites and analogs of possible oxygen intermediates. Present specific aims are to: 1) develop methodology for the study of high-and low-spin FeIII sites which provides the ligand field splitting of the d-orbitals and the covalencies of key ligand-metal bonds; 2) develop L-edge spectroscopy as a probe of differential orbital covalency of non-heme iron sites in relation to heme sites; 3) determine the effects of ligand variation on the FeII and FeIII sites of LO and their contribution to reactivity; 4) determine the role of the FeII site in coupled hydroxylation in the pterin-dependent enzymes; 5) define the nature of the alpha-KG-FeII bond and how this site is affected by different substrates which undergo hydroxylation, oxidative ring closure, or desaturation; 6) determine how the FeII site in the alpha-KG-dependent enzymes correlates to related FeII enzymes which are not alpha-KG-dependent; 7) determine how substrate binding activates the FeII site in the extradiol dioxygenases for O2 reactivity; 8) define the nature of substrate activation by FeIII sites in the intradiol dioxygenases; 9) determine the effects of DNA binding on FeIIBLM, and the electronic structure of its catalytically competent peroxide intermediate; 10) determine the electronic structures of a series of peroxide-FeIII complexes and evaluate their possible contributions to reactivity relative to heme sites.